Desaturase reactions such as the desaturation of oleic acid to linoleic acid, are known to be catalyzed by whole cells of some bacteria, fungi and yeast as a seondary metabolite reaction. These desaturase enzymes require oxygen, cofactors and an electron transport system. Therefore, the reaction using these materials can only be carried out in viable whole cells. Use of desaturase enzymes as in the reaction to convert oleic acid to linoleic acid, has been studied in animals, yeasts, algae, fungi and bacteria. It is known that yeasts tend to possess only mono- and diunsaturated acids of C.sub.16 and C.sub.18, whereas fungi, particularly the lower fungi, contain a rich variety of polyunsaturated fatty acids. The exact composition of the fatty acids produced depends on the relative activities of the various desaturase enzymes, temperature, cell growth rate, and nature of growth-limiting nutrients.
Polyunsaturated fatty acids, for example linoleic acid, which contains 18 carbon atoms and two sets of double bonds, and linolenic acid, which contains 18 carbon atoms and three sets of double bonds, are known to be produced from oleic acid by temperature-induced desaturase enzyme systems. Polyunsaturated fatty acids are almost exclusively incorporated into the cell wall where their low melting point better maintains optimal membrane functions. Psychrophilic yeasts show a higher proportion of polyunsaturates than oleic acid compared to mesophilic yeasts. These findings led to the discovery that the desaturase enzyme synthesis is hyperinduced in bacteria and yeasts initially cultured at 30.degree. C. and shifted to 20.degree. C. The induction of enzyme synthesis occurs within minutes of a temperature shift but is subject to rapid feedback inhibition once the polyunsaturate:saturate ratio in the cell wall has been optimized.
It is further known that oleic acid desaturation rates appear to be the highest among yeasts such as those of the genus Torulopsis, Candida, and Rhodotorula. The desaturation technique is an aerobic process requiring oxygen, ferrous iron, reduced pyridine nucleotides and an electron transport chain. The free fatty acid is not desaturated and olelylphospholipid is the preferred enzyme substrate. Linoleic acid is the principal polyunsaturated fatty acid although it may be further desaturated to linolenic acid if the cells are allowed to age significantly. Nearly all polyunsaturated fatty acids are incorporated into the cell wall in the form of triglycerides, and none are excreted by the cell. The double bonds in the unsaturated acids are in the cis configuration, and where more than one double bond is present, they are normally in methylene-interrupted sequence. The desaturase enzymes have a half-line of ca. 30 minutes at 20.degree. C. and are inhibited by cyanide, azide, metal chelating agents, and reagents that react with sulhydryl or disulfide groups.
Substantial information and work involving these conversion processes, as well as the enzymes and culture mediums for production of the enzymes, are known in the art.
A publication by Yuan, the Journal of Biological Chemistry, Vol. 234, No. 5, May 1961, entitled "Conversion of Oleic Acid to Linoleic Acid" is an important article in this area. This publication discloses conversion of oleic acid to linoleic acid using species of the yeast Torulposis utilis, the specific strain used being ATCC 8205. These authors report that the yeast strain ATCC 8205 of the genus Torulopsis utilis is a lipid-rich yeast known to contain large amounts of linoleate and forms the linoleic acid efficiently from oleate.
U.S. Pat. No. 4,281,064, to Suzuki et al, 1981, discloses a process for producing lipids having a high linoleic acid content wherein fungi of Pellicularia genus are cultivated in a medium of a carbohydrate or vegetable fiber as a carbon source. The culture medium contained glucose NH.sub.4 NO.sub.3, KH.sub.2 PO.sub.4, MgSO.sub.4 7H.sub.2 O, malt extract, yeast extract, FeSO.sub.4 7H.sub.2 O, CaCl.sub.2.2H.sub.2 O, CuSO.sub.4.5H.sub.2 O, and ZnSO.sub.4.7H.sub.2 O in water.
U.S. Pat. No. 3,966,553 to Charpentier et al describes a process for manufacturing citric acid by aerobic culture of yeast strains in medium containing at least one n-paraffin by adding to the culture medium a nitrogenous heterocyclic organic compound. The yeast strains mentioned for use in this process include the genera Torulopsis, Candida, and Rhodotorula. Culture media utilized in this method include those which contain KH.sub.2 PO.sub.4, magnesium sulfate, ammonium nitrate, calcium carbonate, ferric sulfate, manganese sulfate, yeast extract, and water. Similar disclosures are found in published European Application No. 062,492, published Oct. 13, 1982, wherein hydrocarbons are converted to oxidized derivatives by contacting the hydrocarbons with a species of Acinetobacter. In U. K. Patent Application Nos. 2,091,285A and 2,091,286A, published July 28, 1982, methods for the production of fats and oils are described wherein the fats and oils are synthesized using yeast cells capable of synthesizing the fats and oils in a growth medium containing carbon and nitrogen nutrients. Yeast strains of the genus Endomyces, Rhodotorula, Lipomyces and Rhodosporidium are mentioned. In Pat. No. 2,091,285A, culture media which contain a fatty acid in combination with KH.sub.2 PO.sub.4, an emulsifier and antibiotic in water, are disclosed.
U.S. Pat. No. 3,115,442 to Wallen discloses the microbial production of 10-hydroxy-stearic acid using a culture medium containing yeast extract, potassium acid phosphate, magnesium sulfate and water, to which oleic acid is added. The microbe is a species of Pseudomonas. A similar disclosure is found in U.S. Pat. No. 3,975,234, which describes the enzymatic fermentation of a hydrocarbon or alcohol to produce a dicarboxylic acid using a mutant strain of the microorganism Torulopsis bombicola which has been grown on a hydrocarbon free media.
U.S. Pat. Nos. 3,709,783, 3,764,473, and 3,359,177 describe culture media and fermentation methods which include antibiotics for the production of nitrogen compounds such as proteins in U.S. Pat. No. 3,709,783, glutamic acid in U.S. Pat. No. 3,764,473 and nucleotides in U.S. Pat. No. 3,359,177. U.S. Pat. No. 4,340,671 to Gibson discloses an E. coli sensitivity broth which contains antibiotics.
U.S. Pat. No. 3,619,372 to Yoshida et al discloses a lipase which is acid resistant and comprises glutamic acid or similar material. The lipase is prepared by culturing Torulopsis ernobii ATCC 20000 in a liquid medium containing carbon, nitrogen and inorganic salts under an aerobic condition. Vegetable oils or fatty acids may be added to the medium, including oleic acid.
U.S. Pat. No. 3,892,629 describes processes for growing a fungus and producing critic acid using the fungus, Aspergillus niger, as well as other fungi, including species of Penicillium.
In a publication by Difco Laboratories, entitled "Difco Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Procedures", Tenth Edition, pps. 1135-1141, there are set forth a series of culture media for use in the study of yeasts and molds. Included in this study are broths which contain nitrates, phosphates, sulfates and the like, which are identified as Czapek Dox Broth and Czapek Solution Agar on page 257. On pages 1120-1123 is listed W. L. Nutrient Medium which includes yeast extracts, bacto-dextrose, salts of potassium, calcium and magnesium and iron, and manganese, as well as bacto-agar. Various other culture media are disclosed.
While substantial work has been done in this area, it is clear that a need remains in the art to provide methods and systems for isolating and culturing improved desaturase yeast strains which will provide greater yields of the desired products from reactions of this type.